Cell culture method for obtaining prostate-like acini

ABSTRACT

The invention relates to a method for the formation of prostate like acini; acini derived by said method; cells or cell-lines derived from said acini; methods to identify agents capable of inhibiting the proliferation and/or mobility of cancerous prostate cells; agents identified by said method; and methods to identify novel markers of prostate cell differentiation or of cancerous prostate cells.

The invention relates to a method for the formation of prostate acini;acini derived by said method; cells or cell-lines derived from saidacini; methods to identify agents capable of inhibiting theproliferation and/or motility of cancerous prostate cells; agentsidentified by said method; and methods to identify novel markers ofprostate cell differentiation or of cancerous prostate cells.

Prostate cancer is a leading cause of cancer related deaths in men. Theprostate is a male sex gland located in the lower pelvis just below thebladder, the prostate surrounds the urethra, producing the fluidcomponent of semen and helping to control the flow of urine. Enlargementof the prostate is common with age and is a non-maligant condition.Symptoms include, blood in the semen or the urine, frequent pain orstiffness in the lower back, hips or upper thigh. Prostate cancer is adisease of uncontrolled cell proliferation which results in theformation of tumours. The tumours may be primary (ie located in theorgan of origin) or secondary (ie tumours which form in other organs dueto the ability of cancerous cells to move and invade other tissues viathe circulatory system).

Prostate cancer can be relatively harmless or extremely aggressive. Someprostate tumours are slow growing and cause few clinical symptoms.Aggressive prostate tumours spread rapidly to the lymph nodes and otherorgans, especially bone. It is known that the growth of prostate cancercan be inhibited by blocking the supply of male hormones such astestosterone. However, prostate cancers eventually develop and becomeindependent of male sex hormones (ie they become androgen-independentprostate cancer cells). These cells are linked with aggressive,malignant prostate cancer.

All male mammals have a prostate gland but only humans and dogs areknown to naturally develop prostate cancer.

There are a number of model systems which purport to be useful modelsfor the study of prostate cell biology. U.S. Pat. No. 5,874,305describes a prostate cell-line which grows in monolayer. The cell-lineis androgen-independent (not sensitive to the addition of male sexhormones) prostate cancer cell-line. These cells are known in the artand correlate with aggressive tumour forming ability. The cell-line isgrown in monolayer and therefore is not an authentic representation ofthe development of prostate tumours. It would be desirable to targetprostate cancer cells before they become androgen independent and morerecalcitrant to therapy.

In men over the age of 40 the prostate represents a major medicalproblem since both benign prostatic hyperplasia and prostatic carcinomaare becoming increasingly prevalent (Boyle. 1994)). Both the epitheliaand stroma play roles in the advancement of these diseases thereforegood models to study the interaction of these cell types is veryimportant. For example, U.S. Pat. No. 5,917,124 and U.S. Pat. No.5,907,078 disclose transgenic murine models of prostate cancer. Eachpatent discloses the use of prostate specific promoters to driveexpression of SV40 T antigen in transgenic mice. The prostate cellsbecome transformed to reflect the aggressive form of prostate cancer.These transgenic animals do not necessarily reflect the development of aprostate tumour since not all tumours are virally induced. Also, as withstudying androgen-repressed prostate tumour cells, the transgenic modelsonly reflect the aggressive prostate tumours of late stage prostatecancer.

Although animal models exist to study prostatic growth anddifferentiation (Timms, Lee, Aumüller, & Seitz. 1995)) there are nohuman three dimensional models which successfully employ stromal andepithelial cultures to produce morphological and functionaldifferentiation of the prostate. Such models are essential to understandthe normal physiology of the prostate and better understand diseasedevelopment.

In the prostate, rat epithelial cultures have been grown within bothcollagen gels (Ma, Fujiyama, Masaki, & Sugihara. 1997) and Matrigel™(Freeman, Bagli, Lamb, et al. 1994). In collagen, these formedacinus-like structures which secreted Prostate Specific Antigen whilstin Matrigel, spheroids of cell masses with no normal morphological andfunctional differentiation were produced. Normal human prostaticepithelial cell lines cultured in Matrigel showed acinus-like structureswith lumen and PSA secretion (Webber, Bello, Kleinman, & Hoffinan.1997), however, primary cultures formed solid cell masses with littlefunctional differentiation (Hudson, O'Hare, Watt, & Masters. 2000).

We have developed an in vitro cell culture method which provides aculture regime which allows prostate epithelial cells to formprostate-like-acini which closely resemble prostate acini found in vivo.Our method relies on a combination of serum, hormones and a suitablecell matrix support which allows the epithelial cells to attach,proliferate, differentiate and form prostatic-like-acini. The system isable to support growth of cloned normal prostate epithelial cells aswell as cloned cancerous prostate cells, primary prostate epithelialcells, extends life span of cells such as P4, E6 and primary cancerousprostate cells to provide a 3D structure which reflects the in vivostate. The system is invaluable for the study of prostate celldifferentiation and prostate cell transformation. It will provide a toolfor use in the identification of agents effective at inhibiting theproliferation and metastasis of prostate cancer cells and also toidentify novel markers of prostate cell differentiation andtransformation.

The invention relates to a cell culture method which combines a cellsupport matrix to which prostate cells attach and proliferate andculture conditions which combine serum, stromal cell extract andhormones the combination of which promotes the formation of prostatelike—acini with similar characteristics to in vivo acini.

According to a first aspect of the invention, there is provided an invitro method for the formation of prostate-like acini comprising:

-   -   i) providing a cell culture vessel comprising:        -   a) prostate derived cells;        -   b) a cell culture support matrix to which the cells in (a)            can attach and proliferate;        -   c) cell culture medium supplemented serum, a stromal            fraction and a suitable ratio of the hormones oestrogen and            dihydrotestosterone, or functional derivatives thereof;    -   ii) providing conditions which promote the growth and        differentiation of said prostate derived cells in said vessel.

“Vessel” is defined as any means suitable to contain the above describedcell culture. Typically, examples of such a vessel is a petri dish; cellculture bottle or flask; multiwell culture dishes.

In a further preferred method of the invention the supplemented cellculture medium comprises a mixture of serum, stromal fraction, hormonesand cell culture medium to which the prostate derived cells are added.Alternatively, or preferably, the stromal fraction is provided in aseparate vessel, but in liquid contact with the other components of thesupplemented cell culture medium. Typically the separate vessel is aninsert or similar means which allows the cells contained in the stomalfraction to proliferate but prevents cell contact with the prostatederived cells contained in the vessel.

We have observed that a combination of components are required tomaximise acini formation. In the presence of stroma, the number ofspheroids formed approximately doubled and further increased with theaddition of oestrogen and dihydrotestosterone. In addition, presentationof the stroma in the co-culture was examined by comparing stroma withinan insert to that directly mixed with epithelia in a cell culturesupport (eg Matrigel), or added to the top of a preset gel. Our resultsindicated that stroma co-cultured within an insert produced maximalspheroid formation.

In a preferred method of the invention said prostate cells areepithelial cells, preferably human epithelial cells. Preferably saidepthelial cells are derived from prostate glands which have beenmaintained as explants for at least 7 days.

We have observed that the use of epithelial cells from 7 day explantsrather than freshly isolated led to greater acini forming efficiency andthat the acini subsequently produced maintained in culture for longerperiods.

In a preferred method of the invention, the prostate derived cells arenormal (ie non-cancerous), preferably epithelial cells. Normal prostateepithelial cells exhibit number of characteristics for example, thecells are differentiated, have low motility and are non-invasive.Differentiated prostate epithelial cells express a number ofcharacteristic cell markers for example, CK₈, PSA, PSMA, E cadherin.

In a further preferred method of the invention said epithelial cells areprimary prostate epithelial cells.

Current methods which use cell-lines in monolayers typically usecell-lines isolated from one patient. This means any results producedfrom the cell-line are not representative of the male population, northe clonal nature of prostate cancer itself. It is currently believedthat cancer treatments will have to be tailored to an individual'sgenetic profile. Therefore, cell-line models will have limited use inanswering many questions required for clinical diagnosis and devising anappropriate treatment regime for cancer sufferers. Primary culturesprovide a heterogeneous mixture of prostate epithelial cells and arederived from many patients thus providing a model which isrepresentative of cell and patient populations, however, each primarycell line is specific to each patient.

In a further preferred method of the invention said prostate epithelialcells are cancerous.

Cancerous prostate epithelial cells are characterised by anchorageindependent growth, an invasive and motile phenotype. Some cancerousepithelial cells are also characterised by an undifferentiated statewhich is reflected in the lack of expression of cell markers typical ofnormal epithelial cells. Cancerous cells also express a number of uniquecancer specific antigens, so called tumour rejection antigens.

In a further method of the invention there are provided prostateepithelial cells characterised in that said cells are geneticallyengineered by recombinant techniques.

For example, and not by way of limitation, pro-drug activating genes maybe transfected into prostatic cells to monitor the efficacy of pro-drugsas cytotoxic agents. A pro-drug activating gene refers to a gene theexpression of which results in the production of protein capable ofconverting a non-therapeutic compound into a therapeutic compound, whichrenders the cell susceptible to killing by external factors or causes atoxic condition in the cell. An example of a prodrug activating gene isthe cytosine deaminase gene. Cytosine deaminase converts5-fluorocytosine to 5-fluorouracil, a potent antitumor agent. The lysisof the tumor cell provides a localized burst of cytosine deaminasecapable of converting 5FC to 5FU at the localized point of the tumorresulting in the killing of many surrounding tumor cells. This resultsin the killing of a large number of tumor cells without the necessity ofinfecting these cells with a vector (the so-called “bystander effect”).Another example of a prodrug-activating gene is thymidine kinase (TK)(see U.S. Pat. No. 5,631,236 and U.S. Pat. No. 5,601,818) in which thecells expressing the TK gene product are susceptible to selectivekilling by the administration of gancyclovir. This is merely meant to beillustrative of recombinant methods which could be used in combinationwith the cells according to the invention. Other examples may includethe transfection of tumour suppressor genes, (eg p53). The term tumorsuppressor gene refers to a nucleotide sequence, the expression of whichin a target cell is capable of suppressing the cancerous phenotypeand/or inducing apoptosis.

Genetically engineered prostate epithelial cells may be normal primarycells, cancerous primary cells, cloned normal cells or cloned cancerouscells.

In a further preferred embodiment of the invention said prostateepithelial cells are transformed with an oncogene, preferably a viraloncogene (e.g. the HPV E6 or E7 oncogenes, SV40 T antigen).

Methods to introduce nucleic acid into cells are well known in the artand typically involve the use of chemical reagents, cationic lipids orphysical methods. Chemical methods which facilitate the uptake of DNA bycells include the use of DEAE-Dextran (Vaheri and Pagano Science 175:p434). DEAE-dextran is a negatively charged cation which associates andintroduces the DNA into cells but which can result in loss of cellviability. Calcium phosphate is also a commonly used chemical agentwhich when co-precipitated with DNA introduces the DNA into cells(Graham et al Virology (1973) 52: p456).

The use of cationic lipids (eg liposomes, see Felgner (1987)Proc.Natl.Acad.Sci USA, 84:p7413) has become a common method since itdoes not have the degree of toxicity shown by the above describedchemical methods. The cationic head of the lipid associates with thenegatively charged nucleic acid backbone of the DNA to be introduced.The lipid/DNA complex associates with the cell membrane and fuses withthe cell to introduce the associated DNA into the cell. Liposomemediated DNA transfer has several advantages over existing methods. Forexample, cells which are recalcitrant to traditional chemical methodsare more easily transfected using liposome mediated transfer.

More recently still, physical methods to introduce DNA have becomeeffective means to reproducibly transfect cells. Direct microinjectionis one such method which can deliver DNA directly to the nucleus of acell (Capecchi (1980) Cell, 22:p479). This allows the analysis of singlecell transfectants. Electroporation is arguably the most popular methodto transfect DNA. The method involves the use of a high voltageelectrical charge to momentarily permeabilise cell membranes making thempermeable to macromolecular complexes. However physical methods tointroduce DNA do result in considerable loss of cell viability due tointracellular damage. These methods therefore require extensiveoptimisation and also require expensive equipment.

More recently still a method termed immunoporation has become arecognised techinque for the introduction of nucleic acid into cells,(see Bildirici et al, Nature 405, 769) The technique involves the use ofbeads coated with an antibody to a specific receptor. The transfectionmixture includes nucleic acid, typically vector DNA, antibody coatedbeads and cells expressing a specific cell surface receptor. The coatedbeads bind the cell surface receptor and when a shear force is appliedto the cells the beads are stripped from the cell surface. During beadremoval a transient hole is created through which nucleic acid and/orother biological molecules can enter. Transfection efficiency of between40-50% is achievable depending on the nucleic acid used.

In a further preferred method of the invention said cell culture supportis collagen based.

In a yet further preferred embodiment the serum is provided at betweenabout 0.5%-4% (v/v). Preferably said serum is provided at about between1%-3% (v/v). Most preferably said serum is provided at about 2% (v/v).

In a further preferred method of the invention oestrogen is provided atabout 10 ng/ml and dihydrotestosterone at about 10⁻⁷M.

According to a further aspect of the invention there is provided a cellculture composition comprising a collagen based cell support; stroma,oestrogen and dihydrotestosterone.

In a further preferred embodiment of the invention oestrogen is providedat about 10 ng/ml and dihydrotestosterone at about 10⁻⁷M.

According to a further aspect of the invention there is provided aprostate like-acinus formed by the method according to the invention.

According to a yet further aspect of the invention there is provided aprostate like-acinus which has been genetically modified by recombinanttechniques.

According to a yet further aspect of the invention, there is provided acell or cell-line derived from the prostate acinus formed by the methodof the invention. The cell or cell-lines may be genetically engineered.

According to a further aspect of the invention there is provided amethod to identify agents capable of inhibiting the proliferation ofcancerous prostatic cells comprising:

-   -   i) providing culture conditions and at least one cancerous        acinus according to the invention;    -   ii) adding at least one agent to be tested; and    -   iii) monitoring the anti-proliferative activity of the agent        with respect to the cells comprising the cancerous acinus.

According to a yet further aspect of the invention there is provided amethod to identify agents capable of inhibiting the motility ofcancerous prostatic cells comprising:

-   -   i) providing culture conditions and at least one cancerous        acinus according to the invention;    -   ii) adding at least one agent to be tested; and    -   iii) monitoring the motility of cells comprising the cancerous        acinus.

According to a yet further aspect of the invention, there is provided anagent identified by the methods according to the invention.

According to a further aspect of the invention there is provided amethod to identify markers of prostate cell differentiation.

According to a further aspect of the invention there is provided amethod to identify markers of prostate cell transformation.

Methods used in the identification of cell differentiation markersand/or markers of prostate cell transformation include immunogenic basedtechniques (eg using the cells as complex immunogens to develop antiserato cell surface markers and the like) nucleic acid based techniques (egdifferential screeing using cDNA from normal and transformed acini).

Also, it has been known for many years that tumour cells produce anumber of tumour cell specific antigens, some of which are presented atthe tumour cell surface. These are generally referred to as tumourrejection antigens and are derived from larger polypeptides referred toas tumour rejection antigen precursors. Tumour rejection antigens arepresented via HLA's to the immune system. The immune system recognisesthese molecules as foreign and naturally selects and destroys cellsexpressing these antigens. If a transformed cell escapes detection andbecomes established a tumour develops. Vaccines have been developedbased on dominant tumour rejection antigens to provide individuals witha preformed defence to the establishment of a tumour. The methodaccording to the invention provides a means to identify tumour rejectionantigens and precursors which will have utility with respect to thevaccine development to provoke the patients own immune system to deterthe establishment of prostate tumours.

According to a yet further aspect of the invention there is provided anin vitro method to analyse the development of cancerous prostatic cellsfrom normal prostatic cells comprising exposing acini formed by themethod of the invention to at least one agent capable of inducingprostatic cell transformation.

In a preferred method of the invention said normal prostatic cells aretransformed with an oncogene, preferably a viral oncogene.

It is well known in the art that there are agents capable oftransforming a normal cell into a transformed cell with many of thefeatures of cancerous cells. These include, by example only, viruses,DNA intercalating agents, oncogenes, telomerase genes. An embodimentdescribed in the present application is the introduction of the E6 geneusing retroviruses (amphotrophic).

According to a further aspect of the invention there is provided atransformed prostate derived cell wherein said transformation ismediated by a nucleic acid molecule comprising a retroviral vector whichincludes a nucleic acid molecule which encodes a viral oncogene.

Preferably said transformed prostate derived cell is an prostateepithelial cell.

In a preferred embodiment of the invention said retroviral vector is anamphotrophic retrovirus.

In a further preferred embodiment of the invention said oncogene is ahuman papilloma virus oncogene, preferably the E6 or E7 oncogene.Preferably said human papilloma virus is HPV16 and said oncogene is E6.

According to a further aspect of the invention there is provided aretroviral vector wherein said vector includes an human papilloma virusoncogene. Preferably said oncogene is the E6 or E7 oncogene.

According to a further aspect of the invention there is provided amethod to transform a prostate derived cell comprising the steps of:

-   -   i) providing a cell sample comprising prostate derived cells;    -   ii) providing a vector according to the invention;    -   iii) forming a preparation of (i) and (ii);    -   iv) providing transformation conditions wherein said prostate        derived cells are transformed.

In a preferred method of the invention said prostate derived cells areprostate epithelial cells.

An embodiment of the invention will now be described by example, onlyand with reference to the following figures;

FIG. 1. illustrates prostate epithelial spheroids grown in Matrigel andKSFM (sample C). a) Phase image. Bar indicates 80 μm. b) TEM of a wholespheroid, bar indicates 10 μm. c) High magnification TEM indicating bothtight (TJ) and desmosomal-like (D) junctions present between the tightlyassociating inner cells. Bar indicates 1 μm;

FIG. 2. illustrates prostate epithelial spheroids grown in Matrigel andK2 (sample C). a) Phase image. Bar indicates 90 μm b) TEM of a wholespheroid, bar indicates 10 μm. c) High magnification TEM of a whole cellwithin the spheroid, showing luminal microvilli (mv), secretory vesicles(sv) and Golgi apparatus (G). Bar indicates 10 μm;

FIG. 3. illustrates prostate epithelial spheroids grown in Matrigel, K2,10⁻⁷ M DHT, 10 ng/ml OES and stroma (sample C). a) Phase image. Barindicates 70 μm. b) TEM of whole spheroid, bar indicates 10 μm. c) Highmagnification TEM of a whole cell within the spheroid. Secretoryvesicles (sv) are all polarised towards the lumen and microvilli arealso visible on the luminal surface. Bar indicates 6 μm. d) Highmagnification TEM showing the luminal half of an epithelium (shown inc). The figure shows a large active golgi (g) and stacked roughendoplasmic reticulum. In addition a tight junction (TJ) is visible atthe luminal surface. Bar indicates 2 μm. e) A desmosomal-like junctionalcomplex (D) present at a cell:cell interface on a luminal edge. Barindicates 1 μm. f) Basal edge of spheroid showing that no intact basallamina was visible. Bar indicates 1 μm;

FIG. 4 illustrates examples of a budding spheroid with multiple aciniand duct-like structures also with evidence of budding, in phasecontrast (bars indicate 100 μm). Toluidene blue stained thick sectionsof budding and duct-like structures, showing the presence of stratifiedcells (bars indicate 50 μm);

FIG. 5. illustrates stromal cultures increased spheroid formingefficiency. Epithelial sample C was mixed into Matrigel and grown fortwo weeks in either KSFM, K2, K2 and primary stroma (S) or K2, S and10⁻⁷ M dihydrotestosterone (D) and 10 ng/ml oestrogen (O);

FIG. 6. illustrates stromal cultures affect spheroid size. Epithelialsample J was mixed into Matrigel and grown for one week in either K2 orK2 plus primary stroma (S), 10⁻⁷ M dihydrotestosterone (D) and 10 ng/mloestrogen (O) or K2 plus STO cells, D and O. Spheroid size was measuresusing a graticule;

FIG. 7. illustrates dual immunostaining of cytokeratin 18 (green) andcytokeratins 1, 5, 10, 14 (red) of prostatic epithelia grown inMatrigel. Epithelia (sample C) were grown in the presence of KSFM, K2 orK2 plus primary stroma (S), 10⁻⁷ M dihydrotestosterone (D) and 10 ng/mloestrogen (O), for 2 weeks. Cell nuclei in spheroids were counterstained with DAPI (blue). Bar indicates 80 μm.;

FIG. 8. illustrates polarisation of PSA and β1 integrin in Matrigelepithelial spheroids when co-cultured with stroma. Using confocalanalysis the expression of PSA and β1 integrin was compared betweenepithelial spheroids (sample C) grown in K2 or K2 plus primary stroma(S), 10⁻⁷ M dihydrotestosterone (D) and 10 ng/ml oestrogen (O), for 2weeks. Bar indicates 70 μm;

FIG. 9. illustrates examples of immunohistochemical staining ofprostatic epithelial matrigel spheroids. All spheroids shown were grownin KSFM, except for that illustrating androgen receptor expression whichwas cultured in the presence of K2, 10⁻⁷M dihydrotestosterone (DHT), 10ng/ml oestrogen (O) and primary stroma. Spheroid nuclei were counterstained with DAPI (blue). (Epithelial sample C was cultured for 2weeks). Bar indicates 80 μm;

FIG. 10. illustration of prostatic epithelial and stromal cellco-culture in Matrigel;

FIG. 11 illustrates the morphology of Shmac cell lines and P4E6 growingas monolayers. Phase contrast pictures were taken at ×10 objectivemagnification;

FIG. 12 illustrates the growth curves of prostate cell lines growing inK2 medium in monolayer;

FIG. 13 illustrates the invasive ability of Shmac cell lines throughMatrigel coated cell inserts, in response to co-culture with stromalcell lines. Results are expressed as the mean of three triplicates;

FIG. 14 illustrates immunocytochemical staining of Shmac 5 cells growingin monolayer culture A) Dual staining of cytokeratin 18 (red) andcytokeratins 1,5,10,14 (green). B) Vimentin. C) PSA. D) PSMA. E)Androgen receptor. F) E-cadherin. G) β1 integrin. H) CD44. Cell nucleiwere counterstained with DAPI (blue). All images were captured at ×20objective magnification;

FIG. 15: illustrates how stromal co-culture affects spheroids formingefficiency. Epithelial cells were plated into Matrigel and grown for 1week in K2 with (white) or without (black) stromal co-culture. The meannumber of spheroids were counted per field. SE were less than 10% of themean;

FIG. 16 illustrates typical phase contrast morphologies (A) and 1 μmsections (B) of prostate cell line spheroids grown in Matrigel. Allpictures were taken at ×10 objective magnification after 7-10 daysgrowth. Bar, 100 μm; and

FIG. 17 illustrates transmission electron microscopy of Shmac 5epithelial cells grown in Matrigel in the presence of stroma. Cells arecolumnar in shape and show polarization of cellular organelles.Microvilli (mv), secretory vesicles (sv) and Golgi (G) were all luminalwhilst the nucleus (n) was basal. Bar, 2 μm;

FIG. 18 illustrates immunocytochemical staining of Shmac 5 cells growingin Matrigel culture. A) Dual staining of cytokeratin 18 (red) andcytokeratins 1,5,10,14 (green). B) PSA; C) Androgen receptor. D) CD44.E) β1 integrin. Cell nuclei were counterstained with DAPI (blue). Allimages were captured at ×20 objective magnification;

FIG. 19 illustrates Comparative morphology of primary epithelialoutgrowth and the E6 immortalised culture. Shown on the left panel is anepithelial outgrowth from a fragment of prostate tissue. The tissue isthe large black object at the top right of the panel. In the rightpanel, the epithelial component from this outgrowth has been infectedwith a recombinant E6-expressing retrovirus and a cloned epithelialculture produced. Note the similar morphology;

FIG. 20 illustrates Detection of E6 DNA and mRNA in the immortalisedcultures by RT-PCR Agarose gel electrophoresis of PCR products from anE6-transformed prostatic epithelial cell. Marker lane (M) is a 100 bpladder from Life Technologies. Lane 1 is the amplification of cDNA fromthe cell line showing both E6 and E6*-specific products. Lane 2 is anegative control; lane 3 contains DNA from the same cell line (455 bpproduct only) and lane 4 is the CaSki cell DNA positive control; and

FIG. 21 illustrates immunodetection of E6 protein in E6 transformedprostatic epithelial cells Panel A shows positive immunostaining (mainlypancellular) with an anti-E6 antisera (20) of the same cell line asanalysed for DNA and RNA as shown in FIG. 2. Panel B is thecorresponding negative control in which the primary antibody has beenreplaced with PBS in the full staining procedure;

MATERIALS AND METHODS

General chemicals were purchased from Sigma (Poole, UK), tissue culturemedia from Life Technologies (Paisley, UK) and tissue culture plasticfrom (Corning Costar Ltd., High Wycombe, UK) unless otherwise stated.Antibodies were purchased from Dako (High Wycombe, UK) unless stated.

Cell Line Culture

STO cells (mouse embryonic fibroblasts) were obtained from the EuropeanCollection of Animal Cell Cultures (Porton Down, UK) and were routinelycultured in DMEM culture media (Life Technologies, Paisley, UK)supplemented with 10% foetal calf serum (PAA Laboratories, GmbH, Linz,Austria) and 2 mM glutamine (Life Technologies). Cells were routinelycultured without antibiotics in a humidified atmosphere at 37° C. and 5%CO₂.

Prostate Tissue Collection

Non-malignant tissue was obtained from consenting patients undergoingtransurethral resection for benign prostatic hyperplasia orcystoprostatectomy for bladder cancer. 7 samples were collected forepithelial culture (age range 54-86) and 5 for stromal cultures (agerange 57-89), summarised in table 1.

Prostate Primary Cell Culture

Epithelial and stromal cultures were prepared (Lang, Clarke, George,Allen, & Testa 1998) and characterised (Lang, Stower, & Maitland. 2000)as described before, these methods were based on those by Chaproniereand McKeehan (45). Briefly, prostatic tissue was digested by collagenaseand trypsin, and differential centrifugation was used to enrich forepithelial and stromal fractions. The enriched stromal fraction wasresuspended in stromal cell growth medium (RPMI 1640 medium supplementedwith 10% FCS and 1% antibiotic/antimycotic solution) and culturedroutinely in 75 ml tissue culture flasks. Stromal cultures were usedbetween passages 2-5. The epithelial fraction was resuspended inkeratinocyte serum free medium supplemented with 5 ng/ml epidermalgrowth factor, 50 μg/ml bovine pituitary extract and 1%antibiotic/antimycotic solution (media subsequently referred to as KSFM)and passed through a cell sieve (40 μm) to obtain single cells. Singlecells were used immediately for further experiments, frozen for storageor plated into 25 ml flasks in 8 ml of KSFM and grown for 1 week.

Media conditioned by stroma was collected from confluent cultures ofstromal cells by incubating the cultures for 48 hours in 15 ml ofserum-free medium (DMEM/F12 supplemented with 10 μg/ml insulin, 5 μg/mltransferrin and 1 ng/ml selenium). Conditioned medium was removed,filtered (0.2 μm pore) and frozen at −20° C. until required.

Cell Cultures and Viruses

The PA317 murine packaging cell line was (ATCC CRL-9078) was obtainedfrom the American Tissue Culture Collection. Retroviral transfer vectorspLNCX and pLXSN are can be obtained as part of the RetroX kit marketedby Clontech.

Setting up Primary Prostate Epithelial Cultures for RetroviralTransduction

This method has been optimised for prostatic epithelium, but any methodof tissue disaggregation can be employed. A critical step for theamphotrophic retroviral procedure is to obtain dividing cell cultures,as the viral life cycle is not completed in G0 cells (in this case alentiviral vector could be substituted).

Preparation of Biopsy for Culture

Tissue is mechanically disaggregated (chopped) in a sterile petri dishto produce pieces 1 mm² in diameter in 1 ml of transport medium (RPMI1640, 3% (v/v) horse serum, 50 μgml⁻¹ gentamycin (Sigma), 2.5 μgml⁻¹Fungizone).

Seeding of Biopsy Material in Explant Culture

Using a disposable transfer pipette, the disaggregated biopsy isaspirated and transferred to 25 cm² tissue culture flasks with 0.2 μmvented lids (Corning). The medium of choice for the primary culture isdescribed elsewhere (Primary Culture medium). The majority of thedisaggregated tissue specimens should be seeded directly onto tissueculture plastic, but other substrata such as polylysine and collagen canbe used to aid adhesion of the explants.

Maintenance and Growth of Recombinant Amphotrophic Retroviruses.

To generate producer cell lines for retrovirus, the recombinant DNAtransfer vector must be transfected into the packaging cell line (PA317). This requires the insertion of the immortalising gene into atransfer plasmid vector, manipulated in bacteria, which mimics theproviral form of the retrovirus in its most primitive form i.e. atransgene coding region, flanked by the viral LTR sequences. Many suchtransfer vectors exist, and the immortalising gene used in this exampleis inserted in pLXSN (Genbank accession number M28248). This vector alsocontains an SV40 promoter-driven neomycin/G418 resistance gene to allowselection of the producer cells. The immortalising gene (the E6 genefrom human papillomavirus) is under the control of the retroviralpromoter in the LTR. A more elegant (and ultimately safer alternative)is to use the related pLNCX transfer vector (Genbank accession numberM28247) in which the retroviral promoter in the LTR is inactive and theimmortalising gene is under the control of a separate but strongercytomegalovirus immediate early promoter. Complete kits for thegeneration and manipulation of amphotrophic retroviruses are nowavailable commercially (Retro-X from Clontech).

Maintenance of Retroviral Producer Cell Line

The murine fibroblast cell line PA317 is one of several effective hostsfor recombinant retroviruses. It contains the gag, pol and env openreading frames from the transfer vector pMAM3 co-transfected into 3T3cells with an HSV1TK gene. The gag, pol and env genes are constitutivelyexpressed and provide the “packaging” function for any small RNA (<9 kb)with appropriate packaging signals derived from the retroviral terminalLTR sequences, such as pLXSN.

-   1. The cell line is maintained in D10 medium and is subcultured    1:10-1:40 every 4-5 days, using standard techniques.-   2. Replicate frozen stocks of PA317 are prepared in 40% DMEM, 50%    FCS and 10% DMSO and stored in liquid nitrogen.

Generation and Storage of Recombinant Amphotropic Retroviruses

All transfections into PA317 are performed utilising Dosper®transfection (Roche) reagent.

-   -   1. Adherent cells are passaged 1:2 48 hours before transfection,        and again, 24 hours before transfection at 50-80% confluency.    -   2. One microgram of transfer plasmid DNA is mixed with 3.125 μl        of Dosper reagent and serum-free medium added to about 0.1 ml.        The reaction is incubated at 20 C for 15 minutes.    -   3. Immediately before transfection, 10 volumes of the        appropriate complete medium are added to the transfection        mixture and the complete mixture gently pipetted onto the cells.    -   4. Transfections are carried out at 37° C. for 5-12 hours, after        which the transfection mixture is replaced with fresh complete        medium.    -   5. Transfected cells are incubated for 6-16 hours, after which        the cells are washed once with PBS and 1 ml D10 medium per 25        cm² added for a further 24-48 hours.    -   6. Transiently produced retroviruses are collected by removing        the growth medium and floating cells removed by 0.45 μm disc        filtration (Supor®, Gelman Sciences).    -   7. The retrovirus-containing supernatant can be stored at 4° C.        for up to several months or frozen at −80° C. without further        modifications.

Titration of the Virus Stock

-   -   1. HaCaT cells (ATCC number from (18) ) are plated into a 6-well        tissue culture plate at 25-30% confluency, and left to        completely adhere for 12-24 hours.    -   2. The medium is replaced with 0.7-1 ml fresh DF10 containing 8        μg/ml hexadimethrine bromide (polybrene). 10 μl of serial        diluted retroviral supernatant (1:1, 1:2,1:10, 1:100, 1:1000,        1:10000) is added to the prepared HaCaT cells and incubated for        4-12 hours at 37° C.    -   3. After the transduction is complete, the retroviral        supernatant is removed, the cells are washed several times with        PBS, and maintained for a further 48 hours in complete DF10        medium.    -   4. HaCaT cells are then incubated with selection medium        containing 500 μg/ml G418® (PLNCX or pLXSN series. Selection is        carried out for 10-20 days, changing medium every 3-4 days.    -   5. For visualisation of generated colonies the cells can be        stained with Giemsa's staining solution (BDH): The medium is        removed, cells washed once with PBS and 500 μl Giemsa's staining        solution added to each well. Cells are incubated for 10-20 min        and excess staining solution removed with several washed with        tab water. The fixed cells are air-dried and colonies counted.    -   6. The titre is calculated as follows, and expressed in        colony-forming units (cfu) per millilitre of virus (after (19)).        ${{G418}\text{-}{resistant}\quad{CFU}\text{/}{ml}} = \frac{{{number}\quad{of}\quad{colonies}}\quad}{{retroviral}\quad{dilution}\quad{volume}\quad({ml})}$

Immortalisation of Primary Prostatic Epithelium

The actual immortalisation procedure is an extension of protocol above,used to assay the recombinant viral stock. Prostatic epithelial cellsare notoriously difficult to transfect by conventional precipitation orliposome mediated techniques, but our experience with retrovirusesindicates that the cells are readily infectable with as high anefficiency as most mammalian cell lines.

Infection of the Primary Prostatic Epithelial Cells with RetrovirusStock

For infection of primary prostatic epithelial cells undiluted viralstock is used.

-   -   1. The medium is drawn off from the prostatic cell outgrowths.    -   2. Cells are washed 2×5 min. in PBS prior to addition of 1.5 ml        of virus with polybrene at 8 μgml⁻¹ per 25 cm² flask. Cells are        incubated in the presence of the virus for 2 hours at 37° C., 5%        CO2.    -   3. After this time, the polybrene-containing medium is removed        and the cells washed 2×5 min. with PBS. This is replaced with        fresh D10 medium, and the cells incubated for a further 48 hours        prior to selection with G418 at 25 μgml⁻¹.

Ring Cloning of Transfected Cells

After 10-14 days of drug selection discrete colonies are observed whichcan be individually ring cloned into 12.5 cm² flasks.

-   -   1. Cells are washed 2×3 min. in PBS-Ca²⁺/Mg²⁺, and the lid of        the flask cut off under sterile conditions.    -   2. A sterile glass ring (10 mm in diameter) is dipped in        autoclaved petroleum jelly and placed over the individual        colonies, creating a seal.    -   3. 500 μl of 0.25% (v/v) trypsin/EDTA was placed in the ring and        immediately aspirated off together with the PBS.    -   4. Trypsin is again applied and the cells monitored. As they        began to round up, cells are gently pipetted up and down and        placed in flasks containing R10 culture medium.

The isolated colonies are incubated at 37° C., 5% CO₂, until they can besubcultured into larger tissue culture flasks.

Cell Lifts for DNA Purification

To monitor the immortalisation process, and to provide an indication ofthe origin of the cells immortalised, a micro-assay from the growingimmortalised colonies of epithelial cells can be carried out. Using theprocedure described below, sufficient cells are obtained to carry out aPCR amplification of either known genes (to compare mutation statusbetween the original tumour and the cellular outgrowths) or amicrosatellite/Single nucleotide polymorphism analysis. Full protocolsfor the latter analysis are available elsewhere.

-   -   1. The medium is aspirated from growing cells, which are then        washed 2×5 min. in PBS.    -   2. Squares of 3 MM paper are cut (measuring 3×3 mm) and        sterilised by autoclaving in a glass petri dish.    -   3. Using sterile forceps, the 3 MM squares are placed onto the        cells, left for 20 sec. and then removed into an eppendorf tube        containing 200 μl of DNA extraction buffer.    -   4. Fresh medium is restored to the cultured cells, which can        then continue growing.    -   5. The 3 MM squares are processed as follows. Fifty microlitres        (or more) are added to an eppendorf tube containing the 3 MM        square and incubated overnight at 42 C.    -   6. Next day, the proteinase K was inactivated by incubation at        95 C for 8-10 minutes, and the paper either centrifuged to the        bottom of the tube or carefully removed with a sterile tip.    -   7. The resulting solution is ready for PCR amplification and        further analysis, when used to make up no more than 10% of a PCR        reaction final volume.

Detection of E6 DNA in Infected Cells

-   -   1. Cells are pelleted by centrifugation and the DNA extracted by        standard methods. 20 ng of the DNA is used as a substrate for        PCR.    -   2. Reaction mixtures contain 2 mM dNTPs, 0.05% W-1, 1.5 mM        MgC12, 0.3 μM forward and reverse primers (see note 6) and 0.5 U        of Taq. DNA polymerase. Thirty five cycles of amplification,        with annealing at 55 C are sufficient to detect the low copy        numbers of E6 retained in the immortalised cells.    -   3. PCR product (455 bp) is detected by electrophoresis in a 1%        (w/v) agarose gel.

RT-PCR to Detect E6 mRNA

-   -   1. 5 μg total cell RNA is pipetted into a DEPC-treated Eppendorf        tube with 0.5 μl of RNAguard solution (Boeringer Mannheim), 500        ng of oligo-dT primer and sterile ddH₂O to a volume of 10.5 μl.    -   2. The tube is heated to 70° C. for 10 min. and snap-cooled on        ice. The Eppendorf tube is then pulsed in a microfuge to collect        the contents.    -   3. The cDNA synthesis reaction is set up by the addition of 0.5        μl RNAguard, IX Superscript buffer (Boehringer Mannheim), 10 mM        DTT, 1 mM dNTPs and 200 U of Superscript enzyme.    -   4. The contents of the tube are mixed gently and incubated at        42° C. for 1 hr. After this period the cDNA is precipitated at        −80° C. for 1 hr. by addition of 0.05 volumes of glycogen        solution, 0.5 volumes of 3 M NaCl and 3 volumes of absolute        ethanol.    -   5. The cDNA is pelleted by centriftigation at 15,000 rpm at        4° C. for 5 min. and following a final wash with ice-cold 70%        ethanol the pellet is air-dried and resuspended in 20 μl sterile        ddH₂O.    -   6. 2 μl of cDNA is used as a substrate for PCR to detect E6        expression. The reaction mixture also consists of 2 mM dNTPs,        0.05% W-1 detergent, 1.5 mM MgCI₂, 3 pmoles of each of the        forward and reverse primers (see note 6), 1×PCR buffer and 1 U        of Taq. DNA polymerase (Gibco-BRL).    -   7. Products are resolved by electrophoresis in a 1% (w/v)        agarose gel as shown in FIG. 20.

Shmac Cell-Lines

The Shmac series of prostate epithelial cell lines were derived from asequential series of tissue biopsies, grown as explants in primaryculture as described above and infected with E6 retrovirus as describedin Maitland et al (2001). Individual populations selected by drug (G418)resistance are not immortal (like P4E6) but have an extended life span.

Immortalisation and Culture of Cell Lines

Shmac 2, 3 and 6 cells were derived from benign prostatic hyperplasia.Shmac 4 cells were derived from a well differentiated tumour (1+2) andShmac 5 from a moderately differentiated tumour (3+3). P4E6 wasimmortalised from prostate epithelial cells derived from a welldifferentiated tumour, Gleason score 4 [Maitland et al 2001]. Epithelialcell lines were routinely cultured in keratinocyte serum free mediasupplemented with 2% foetal calf serum (PAA Laboratories, GmbH, Linz,Austria), 5 ng/ml epidermal growth factor and 25 μg/ml bovine pituitaryextract (K2).

STO cells (mouse embryonic fibroblast cell line) were obtained from theEuropean Collection of Animal Cell Cultures (Porton Down, UK) and wereroutinely cultured in DMEM culture media supplemented with 10% foetalcalf serum and 2 mM glutamine. All cells were routinely cultured withoutantibiotics in a humidified atmosphere at 37° C. and 5% CO₂.

Cell Morphology and Growth Assay of Shmac Cell-Lines

Phase images were observed with a Nikon TE300 inverted microscope andcaptured with a JVC 3-CCD video camera. Images were subsequentlyprepared using Adobe Photoshop 4. For growth assays cells were preparedat a concentration of 10⁴ cells/ml in appropriate growth media. 200 μlof cell solution was then added to the well of a 96 well plate. Cellswere media changed or counted every 3-4 days. Cell counts were performedby haemocytometer after trypsinisation. Cell solutions were diluted withtrypan blue and viable cell counts taken.

Invasion and Motility Assays for Shmac Cell-lines

Both assays were performed as detailed in Lang et al, 2000. Briefly,motility was measured by observing an epithelial cell colony ofapproximately 8-16 cells. Phase contrast images were captured every 4minutes for 8 hours using a JVC video camera, and recorded on computerusing a Scion Image CG7 frame grabber [Scion Corporation, Frederick,Md., USA]). Motility was scored by assessing membrane ruffling,pseudopodial and translative movement, based on the method of Mohler etal, 1988.

Invasion was measured by counting the number of epithelial cellsinvading Matrigel (Becton Dickinson, Oxford, UK) coated cell cultureinserts (8 μm pore, Becton Dickinson) in serum free medium. Inserts wereplaced in 24 well plates which contained confluent cultures of STOstromal cells. Epithelial invasion was measured overnight, after whichthe inserts were removed and crystal violet was used to stain and countthe cells which had invaded to the underside of the insert.

Isolation of CD44+ Epithelial Cells

Single cell suspensions of primary prostatic epithelia (10⁶ cells) werelabelled with 2.5 82 g anti-CD44 (Pharmingen, Becton Dickinson UK Ltd.,Oxford, UK) for 5 mins at 4° C. and then washed extensively using PBSsupplemented with 2 mM EDTA and 0.5% (w/v) BSA. Antibody was then linkedto 20 μl goat anti-mouse MACS microbeads (Miltenyi Biotec Ltd., Bisley,UK) at 4° C. for 15 mins, the cells were again washed extensively afterwhich they were added to a MACS column and the labelled basal cells wereeluted and resuspended in appropriate culture media (basal cells formed10-43% of the total epithelial cell population).

Cell Culture in Matrigel

Epithelial cells were prepared at a concentration of 60 000 cells/ml inKSFM. On ice they were mixed 1:1 (v/v) with Matrigel (Becton Dickinson,Oxford, UK) and 0.25 ml aliquots were subsequently plated into 24 wellplates. The Matrigel was set by incubating at 37° C. for 30 mins. Forexperiments requiring stromal co-culture, stroma was pre-grown onto cellculture inserts, these were then placed on top of theMatrigel/epithelial cell mix (illustrated in FIG. 1). 1 ml of requiredgrowth media was added to each well and cells were thereafter mediumchanged every 3 days, by the removal of 0.5 ml of spent media and theaddition of 0.5 ml of fresh media. Equivalent batches of Matrigel wereused throughout. Phase images were observed with a Nikon TE300 invertedmicroscope and captured with a JVC 3-CCD video camera. Images weresubsequently prepared using Adobe Photoshop 4.

Transmission Electron Microscopy

Cells growing in Matrigel were washed twice with phosphate bufferedsaline (PBS) and then fixed for 1 hour at room temperature in 100 mMphosphate buffer, 4% paraformaldehyde (TAAB, UK) and 2.5% Ultrapureglutaraldehyde. Cells were further processed for electron microscopy asdescribed by Allen and de Wynter (46). Thick sections were cut at 1 μmand stained with 0.6% toluidene blue in 0.3% sodium bicarbonate. 70 nmsections were cut and stained with saturated uranyl acetate in 50%ethanol followed by Reynolds lead citrate and observed with a Jeol JEM1200 Ex transmission electron microscope.

Fluorescent Immunostaining

Cells grown in Matrigel were snap frozen in liquid nitrogen afterembedding the gel in OCT Compound (BDH, Poole, UK). Embedded gels werestored at −20° C. 7 μm sections were cut on a Leica cryostat and mountedonto Super frost microscope slides (BDH).

Immunostaining was carried out according to table 2. Antibodies wereprepared in PBS supplemented with 1% bovine serum albumin. Each step wasfollowed by three washes in PBS. Primary antibodies were incubated atroom temperature for one hour and secondary antibodies for 30 minutes.Spheroids were counter stained with 1 μg/ml DAPI. Coverslips weremounted to slides using Cityfluor (Agar Scientific Limited, Stansted,UK). Immunostained cultures were observed and photographed using a NikonEclipse TE300 fluorescent microscope. Digital images were subsequentlyprepared using Adobe Photoshop 4.

Confocal Microscopy

Samples embedded in OCT were sectioned at 20 μm and inmmunostained asdescribed above. Sections were then observed at 1 μm layers using aMRC1000 Biorad Confocal Microscope (Hemel Hempstead, UK).

EXAMPLE 1

Seven day cultures of non-malignant prostatic epithelia were seeded assingle cells directly into Matrigel, and in the presence of KSFM, thesingle cells developed into spheroids which were irregular in shape(FIG. 1 a). TEM demonstrated that these spheroids were solid masses ofboth cuboidal and stratified cells and their appearance was consistentwith a hyperplastic growth (FIG. 1 b). In the centre of the spheroidthere was evidence of necrosis. The central stratified/cuboidal cellshad very tight cell to cell contacts (FIG. 1 c) whereas the outercuboidal cells contacted each other much more loosely, had relativelysparse cytoplasms and elongated nuclei with prominent nucleoli. Highpower magnification indicated there were multiple junctional complexesconsistent with desmosomal-like and tight junction-like cell contacts,present between both cell types (examples shown in FIG. 1 c). Thepresence of oestrogen, dihydrotestosterone or media conditioned byprostatic stromal cultures did not affect the morphology (results notshown).

Addition of 2% serum to the media led to the spheroids appearing lessdense (FIG. 2 a), TEM indicated this was due to the spheroids developinglumen (FIG. 2 b). The spheroids had ½ epithelial cell layers and werecuboidal or columnar in shape. Microvilli were observed at the luminaledge of the epithelium but other signs of polarisation were not evident.Golgi bodies, secretory vesicles and stacked rough endoplasmic reticulum(RER) were all present, consistent with a secretory function. No basallamina was observed and few junctional complexes were observed. Serumwas included in these experiments to support stromal growth.

Research has shown that stroma, oestrogen and dihydrotestosterone arerequired to induce prostate epithelial differentiation (Bayne, Donnelly,Chapman, Bollina, Buck, & Habib. 1998)). The addition of these factorsplus serum to epithelium growing in Matrigel led to the formation ofcompact spheroids which were regular in shape (FIG. 3 a). TEMdemonstrated the spheroids were similar to in vivo acini since theycontained lumen surrounded by one or two epithelial cell layers whichwere closely organised and columnar (FIG. 3 b). Higher magnification(FIG. 3 c) indicated the cells were polarised, such that microvilli,golgi and secretory vesicles were organised to the luminal side whilstnuclei were predominantly basal. Golgi were consistently large andstacked RER was evident. No intact basal lamina was visible though agreater number of junctional complexes (desmosome-like and tightjunction-like) were visible laterally and were predominantly toward thelumen.

This experiment was carried out in parallel on three differentepithelial cultures (B, C, D). The results were similar for all, howeveronly sample C (shown in FIGS. 1-3) demonstrated a high degree ofpolarisation in the presence of stroma (FIG. 3 b). Samples B and Cproduced lumen containing spheroids with columnar or cuboidal epitheliumbut no polarisation.

Table 1 summarises all repeat experiments in K2, DHT, Oes and stroma.Overall experiments in these culture conditions showed evidence ofcolumnar polarised epithelia in 2/4 examined epithelial samples (C, J).In two separate experiments spheroids did not grow in serum freeconditions (samples F and G). Growth in 2% serum consistently led to theformation of spheroids with lumen (5/7 samples) where it did not therewas no growth (sample F) or there was irregular spheroid formation(sample G), as illustrated in FIG. 1. The sample showing no growth,produced spheroids only in the presence of stroma and in this instancethe spheroids were irregular. The sample which produced irregularspheroids in 2% serum, produced lumen in the presence of stroma but theepithelia were cuboidal, columnar and stratified, with no evidence ofpolarisation. Subsequently, TEM analysis of polarisation was onlycarried out if compact acinus-like spheroids were observed.

Basal epithelium in the prostate express CD44 (25) and may represent acandidate epithelial population more likely to differentiate in Matrigelculture. Therefore, we selected CD44 positive epithelium from four (ofseven) prostate epithelial preparations (F, G, I, J). No noticeabledifferences were observed in spheroid formation or morphology incomparison to those produced from whole epithelial populations (samplesB, C, D). However, CD44 negative epithelial populations showed no growthwithin Matrigel (results not shown). Two of the samples (B, F) formedbudding and ductal structures when grown in K2, DHT, OES and stroma, afurther two (D, G) also exhibited such morphologies when grown withoutstroma (examples shown in FIG. 4). All samples which produced buddingand ductal structures were accompanied by stratified epithelia. Sinceepithelia are not normally stratified in prostatic duct or acini weconcentrated our studies on the acinus-like spheroids.

EXAMPLE 2

The presence of stromal cultures was found to significantly increase thespheroid forming efficiency of epithelial samples. FIG. 5 indicates thatapproximately equal numbers of spheroids formed in KSFM and K2 (sampleC). In the presence of stroma, the number of spheroids formedapproximately doubled, and further increased with the addition ofoestrogen and dihydrotestosterone. Two other samples (B and D) examinedin parallel, showed increased spheroid formation only in the presence ofstroma (approximately double), but hormones had no further effects.Increased spheroid formation in the presence of stroma was reproduced onthree further samples examined on separate occasions (F, G, J). Inaddition, presentation of the stroma in the co-culture was examined bycomparing stroma within an insert to that directly mixed with epitheliain the Matrigel, or added to the top of a preset gel. Our resultsindicated that stroma co-cultured within an insert produced maximalspheroid formation. In addition, the different ways of presenting stromahad no effect on spheroid morphology (results not shown). We alsoobserved that the use of epithelial cells from 7 day explants ratherthan freshly isolated led to greater spheroid forming efficiency andthat the spheroids subsequently produced maintained in culture forlonger periods (results not shown).

EXAMPLE 3

The size and type (irregular or acinus-like) of epithelial spheroidsforming within Matrigel varied between samples (summarised in table 1).However we consistently observed that stromal co-cultures predominantlyproduced smaller sized spheroids. FIG. 6 shows that, after 1 week inMatrigel and K2, equivalent numbers of 0.1 mm and 0.2 mm diameterepithelial spheroids had grown (22 cells/field did not form spheroidsbut remained as single cells). In total, an average of 30 spheroids/field formed. In the presence of primary and cell line stroma 36 and 43total average spheroids/field, formed respectively, but werepredominantly 0.1 mm in diameter. Notably, co-culture with STO cells ledto greater numbers of spheroids forming.

EXAMPLE 4

Spheroids were sectioned and stained by fluorescenceimmunohistochemistry to compare the phenotypic profiles of those grownin serum free conditions to those grown with sera, stroma and hormones.The spheroids were phenotyped by investigating a variety ofdifferentiation markers. Luminal prostatic epithelium were identifiedusing; cytokeratin 18 and prostate specific antigen (PSA) (Nagle.1996)), whilst basal epithelial cells were identified using; basalcytokeratin (1,5,10,14), CD44 and β1 integrin (Knox, Cress, Clark, etal. 1994)). Vimentin was analysed since it can reflect differentiation(Iwatsuki, Sasaki, Suda, & Itano. 1999). Androgen receptor, PSA andprostate specific membrane antigen (PSMA) served as functionaldifferentiation markers (30, 31). Finally, the cell adhesion molecules,E-cadherin and desmoglein were also analysed. The results are summarisedin table 2 and examples of each stain are shown in FIGS. 7, 8, and 9.Intermediate filaments stained at similar intensities between thedifferent spheroid types (table 2). However, localisation ofcytokeratins 18 and 1,5,10,14 varied between the different spheroids(FIG. 7). Spheroids grown in the presence of KSFM showed expression ofcytokeratins 1,5,10,14 in the epithelia at the outer edge of thespheroid, whilst cytokeratin 18 was expressed independently by the cellsin the middle of the spheroid. Spheroids grown in the presence of serumand/or stroma were predominantly cytokeratin 18 positive but alsoco-localisation of cytokeratins 18 and 1,5,10,14 was observed. PSA wasstrongly expressed in all the spheroids, but, expression was polarised(towards the lumen) in spheroids grown in the presence of stroma (FIG.8). PSMA was strongly expressed by all spheroid types, but expressionwas stronger in the outer cells of spheroids grown in serum freeconditions (FIG. 9). Androgen receptor was only weakly detected inspheroids grown with stroma (FIG. 9). E cadherin and desmoglein wereexpressed by all spheroids at cell to cell contacts. CD44 and β1integrin were likewise strongly expressed by all spheroids at the cellmembrane, but noticeably both markers were only expressed by the outercells of spheroids grown in serum free conditions. In addition, β1integrin expression was strongly polarised (basally) in the presence ofstroma (FIG. 8).

The present study demonstrates for the first time that human primaryprostate epithelium seeded into Matrigel can form acinus-like structuresin the presence of stroma, androgen, oestrogen and serum. These acinishow a high degree of functional (PSA+/PSMA+/androgen receptor+) andmorphological differentiation consistent with human prostatic acini invivo. This represents a very useful model with which to study prostaticbiology and will complement existing animal models (Hayward, Rosen, &Cunha. 1997)) by reducing the complexity inherent in such systems.

PSA expression can be induced by Matrigel alone (shown here) or stromaalone (Bayne, Donnelly, Chapman, Bollina, Buck, & Habib. 1998)).Induction of PSA expression by epithelium without stroma indicates thatepithelial differentiation is partly inherent. In our model, bothMatrigel and stroma were clearly required to induce architecturalorganisation, androgen receptor expression and polarised secretion ofPSA. Previously, androgen receptor expression in human primary prostatehas been observed in both epithelia and stroma when co-cultured togetherbut not in isolation (Bayne, Donnelly, Chapman, Bollina, Buck, & Habib.1998), emphasising the importance of both cell types for terminalepithelial differentiation. The requirement for stroma to induce thecorrect architectural organisation has previously been demonstrated inmouse models (32). Our results also found that stromal co-cultureproduced greater numbers of small spheroids, which may indicate that thestroma and hormones either reduced growth or increased adhesion (therebycompacting the cells into a smaller spheroid). Stroma was clearlyimportant for increasing spheroid forming efficiency. The ability ofstroma to double spheroid forming efficiency suggests that stroma canrecruit more epithelia to form spheroids. It is possible that epitheliumin isolation can form spheroids if they have already received signals todifferentiate but are then unable to undergo proper differentiation. Thefactors governing these differentiation pathways are unknown. Onestromal derived factor, hepatocyte growth factor was found to increasethe growth of primary lung epithelium and also increase the numbers ofspheroids formed in Matrigel two-fold (Sato & Takahashi. 1997).Hepatocyte growth factor is clearly worth further investigation in ourown model system.

The addition of unknown serum factors to Matrigel went some way toproducing the correct morphological organisation of spheroids intoacinus-like structures, but stromal co-culture was required for greaterdifferentiation. Experiments examining the behaviour of primary mammaryepithelium cultured in Matrigel alone found that breast specificproteins can also be expressed (Chen & Bissell. 1989)5). However, breastepithelial spheroids can be grown in Matrigel and demonstrate bothfunctional and morphological differentiation in the presence of serumand hormones alone (Barcellos-Hoff, Aggeler, Ram, & Bissell. 1989).Stromal co-culture systems increased differentiation, and, in agreementwith our own results, produced alveolar morphogenesis rather that ductal(Darcy, Zangani, Shea-Eaton, et al. 2000). Breast studies have shownthat differences in growth factor/ kinase receptor activation canaccount for alveolar or ductal morphologies (Niemann, Brinkmann,Spitzer, et al. 1998). Recent studies indicate that a complex mix ofgrowth factors and hormones can override the need for serum when breastepithelium is grown in Matrigel with stromal co-culture (Darcy, Zangani,Shea-Eaton, et al. 2000), and such studies are clearly now required tounderstand the important factors in prostatic differentiation. Stromalco-culture is also required for the induction of functional andmorphological differentiation in other organ models, such as ovarianepithelium (Ohtake, Katabuchi, Matsuura, & Okamura. 1999). Thedifferentiation of urothelium in collagen matrix is also dependent onthe formation of a basement matrix specifically driven by stromalinteractions, and not by soluble stromal factors (Fujiyama, Masaki, &Sugihara. 1995). The breast and ovarian models discussed above all foundevidence of a complete basal lamina forming beneath the epithelium,whilst our results found only an incomplete basal lamina suggesting thatMatrigel alone was sufficient to induce differentiation, a phenomenonalso reported with rat prostate (Ma, Fujiyama, Masaki, & Sugihara.1997).

Previous attempts to produce the prostatic model described here havefailed to produce morphological differentiation, most likely due to theabsence of stromal cultures. Early attempts using primary rat epithelia(Freeman, Bagli, Lamb, et al. 1994) and more recently human primaryepithelia (Hudson, O'Hare, Watt, & Masters. 2000) both successfully grewspheroids in serum free media and in both instances spheroids of solidcells exhibiting a phenotype of hyperplastic growth were produced. Suchmorphologies were evident even in the presence of soluble stromalfactors and the expression of androgen receptor (Hudson, O'Hare, Watt, &Masters. 2000). In contradiction of this, prostatic cell lines canundergo morphological and functional differentiation in Matrigel whenplated without stroma (Webber, Bello, Kleinman, & Hoffman. 1997),suggesting the immortalisation process can override the requirement forstromal cells to induce full differentiation as described here. Theimportance of mesenchyme for epithelial differentiation is fundamentaland has been demonstrated by numerous animal studies (Timms, Lee,Aumüller, & Seitz. 1995)17). Mesenchyme from different origins caninduce epithelia to differentiate along different pathways. For exampleurogenital mesenchyme can induce bladder epithelium to undergo prostaticdifferentiation, indicating the potential existence of a urogenital stemcell (39). More recently, a greater contribution of stroma towards thedevelopment of disease is being considered. Studies have shown thatstroma from different reproductive states of the of the breast (Bemis &Schedin. 2000) or prostate tumours (Lang, S H., Stower, M. and Maitland,N. J. (2000) In vitro modelling of epithelial and stromal interactionsin non-malignant and malignant prostates. British Journal of Cancer82(4): 990-997, Hall, J., Maitland, N. J., Stower, M., Lang, S., (2001)Primary Prostate Stromal Cells Modulate the Morphology and Migration ofPrimay Prostate Epithelial Cells in Type 1 Collagen Gels. CancerResearch 62: 58-62) can modulate invasion and motility of theepithelium, characteristics clearly important for cancer progression.Our model will provide a useful tool for studying how epithelial/stromalinteractions contribute towards cancer progression.

The formation of spheroids in Matrigel in the presence of serum haddistinct effects on the phenotypic profile of the epithelium. Spheroidsgrown in serum free media showed two distinct cellular compartments. Theouter cells of the spheroid were basal in morphology and phenotype(cytokeratin 1,5,10,14+/cytokeratin 18−/CD44+/β1 integrin+), whilst thecentral cells were intermediate (cytokeratin 1,5,10,14+/cytokeratin18+/CD44−β1 integrin−) or luminal in phenotype (cytokeratin1,5,10,14−/cytokeratin 18+/CD44−/β1 integrin−). The presence of PSA inall the cell populations indicates that the basal-like cells are morelikely early intermediate in phenotype. These serum free spheroids aresimilar to those previously produced by Hudson et al (21) and also tothe budding structures produced in monolayer culture by van Leenders etal (42). Phenotypically the spheroids produced in serum free conditionsare more similar to in vivo acini since they contain separate cellularcompartments (basal and luminal-like layers). The lack of lumen andcolumnar, luminal epithelium means they are morphologically dissimilar.Spheroids grown in the presence of serum, hormones and stroma producedspheroids which are morphologically very similar to in vivo acini.Phenotypically they show intermediate (cytokeratin1,5,10,14+/cytokeratin 18+/PSA+/AR+) or luminal-like (cytokeratin1,5,10,14−/cytokeratin 18+/PSA+/AR+) epithelial profiles but thepresence of a distinct basal layer is lost. The presence of androgenreceptor and a more complete morphology indicates these spheroids aremore differentiated than those grown in serum free conditions. It ispossible that the majority of spheroids (grown under any conditions) arederived from early basal cells and only a few are derived from stem cellpopulations. Those derived from early basal cells would have thecapacity to differentiate but not replace a basal cell population. Thoseelusive spheroids derived from stem cells may therefore contain basaland luminal cells in a morphologically differentiated spheroid. It ishighly likely that a proportion of the primary epithelium used in thesestudies were stem cell-like (or early basal cells) since they wereproliferative and pluripotent (capable of producing both basal andluminal cells, stratified, columnar or cuboidal cells and alsoacinus-like or duct-like structures). Spheroids derived from CD44+(basal) cells certainly gave rise to PSA+/cytokeratin 18+/CD44−(luminal) cells. This study provides further evidence for thehierarchical relationship in which basal and luminal cells are linked ina precursor progeny relationship. The heterogeneous expression ofseveral markers of basal and luminal cells suggest that the putativestem cell population, reside within the basal layer and give rise tointermediate cells (cytokeratin 1,5,10,14+/cytokeratin 18+/PSA+) andterminally differentiated cells (cytokeratin 18+/PSA+/AR+).

Our experiments showed a variation between tissue samples. This is notunexpected given the diversity of tissue samples and the heterogeneousnature of prostatic disease. Indeed this type of analysis will bring uscloser to the phenotype of prostate tumours than the analysis of a fewcell lines. The age of the patient from which the tissue was obtainedalso plays a role in culture. In our experiments (table 1) culture oftissue from 70-90 year olds was less successful than that from youngerpatients (54/57 years old). This may indicate that the model requires aviable stem cell population, since stem cells will be more predominantin younger tissue. Thus our future studies will concentrate on the useof younger tissue whilst trying to analyse the contribution of age andstem cell populations to the formation of acinus-like structures.

EXAMPLE 5 Morphology of Cell Lines in Monolayer

All the cell lines showed a typical epithelial morphology, which wasround or cuboidal and became cobblestone-like when confluent (FIG. 11).Untypically, all cell lines also showed the presence of pseudopodialextensions in a percentage of the cell population, particularly whensubconfluent. Shmac 2 cells were notable for having a stringy andvacuolated appearance. Shmac 3 cells were the largest in appearance.Shmac 4 cells were notable for the appearance of blebs at the cellmembrane.

EXAMPLE 6 Growth in Monolayer

Shmac 3 cells did not grow successfully beyond 3 or 4 passages afterimmortalisation, thus further experiments were not attempted. Shmac 2cells were not used beyond passage 10, whilst Shmac 4,5,6 and P4E6 werenot used beyond passage 15. FIG. 12 indicates the growth of all othercell lines in K2 over a period of 17 days. Overall Shmac 6 and P4E6 grewvery quickly and soon reached confluence. Doubling times were just over24 hours for P4E6 and 48 hours for Shmac 6. Shmac 5 cells grew slowly tostart with a doubling time of approximately 5 days but then grew morequickly to confluence. Shmac 2 and 4 grew very slowly and did not reachconfluence after the 17 day culture period.

EXAMPLE 7 Invasion and Motility of Cell Lines

The potential metastatic ability of the Shmac cells was investigated bymeasuring their motility and invasive ability, in vitro. Shmac 5 was theonly cell line capable of invasion through a Matrigel coated insert.MDA-MB-231 and P4E6 cells were included as positive and negativecontrols respectively. In contrast, all cell lines showed high levels ofmotility, though this was mainly confined to ruffling of the cellmembrane. Shmac 4 cells showed lots of translation as a scatteredcolony, whilst only Shmac 5 and 6 were capable of individual celltranslation. The invasion and motility of P4E6, PNT2-C2 and PC-3 havebeen measured before (Lang et al, 2001) but were included forcomparison.

EXAMPLE 8 Phenotype of Cell Lines in Monolayer

The cellular phenotype of the cell lines was determined byimmunocytochemistry. We examined a standard variety of cellular markers,as previously reported {25970}. Cytokeratins 1,5,10,14, the β1 integrinfamily and CD44 are all markers of basal prostatic epithelium, whilstcytokeratin 18, prostate specific antigen (PSA), prostate specificmembrane antigen (PSMA) and androgen receptor are all markers of luminalor functionally differentiated prostate epithelia. In addition the celladhesion marker, E-cadherin and mesenchymal marker vimentin were alsoinvestigated. The results for all Shmac cells are summarized in table 2(P4E6 are included for comparison) and an example of each stain isdemonstrated for the Shmac 5 cell line in FIG. 14.

All cell lines except Shmac 5 and P4E6 showed very strong cytokeratin 8staining in the cytoplasm. For Shmac 5 and Shmac 2 this staining wasconfined to roughly 50% of the cell population and was seen moststrongly expressed by cells which were uppermost in the culture.Stronger expression was also noted for Shmac 4 and 6 in cells which weresat on other cells in a monolayer culture. Conversely, most cell linesshowed weak basal cytokeratin staining, except Shmac 5 and Shmac 4.Vimentin staining was moderate or strong in all cell lines. PSAexpression was weak in most cell lines except P4E6 and Shmac 4 where itwas moderate. PSMA was moderate to strong in all and was expressed inthe cytoplasm or located to the cell membrane. No androgen receptorexpression was detected in any cell line, though a minority of cells mayhave shown weak expression (see FIG. 14E).The basal markers CD44 and β1integrin were strongly expressed by nearly all the cell lines andstaining was membrane or cell to cell or cytoplasmic. SimilarlyE-cadherin expression was found in all cell lines and the cell to cellexpression indicated the protein was functional.

EXAMPLE 9 Growth and Morphology of Shmac Cell Lines and P4E6 Crown inMatrigel

Previous investigations of the common prostate cell lines found thatonly PC-3 (not PNT2-C2, PNT1a, DU145 and LNCaP) could form spheroids inMatrigel that resemble in vivo acini (Lang et al 2001a and unpublishedresults). To determine what cellular factors are important to establishin vitro acini we examined the wider range of Shmac cell lines and P4E6.Experiments with primary cultures indicated that stromal co-culturecould enhance differentiation of the in vitro acini therefore cells wereplated with and without stromal co-culture.

Shmac 4 cells did not form spheroids large enough to form lumen after 14days in Matrigel culture. Stromal co-culture increased the number ofspheroids forming from the culture of Shmac 5, 6 and P4E6 (FIG. 15), buthad little effect on spheroid formation from Shmac 2 cells. FIGS. 16 aand 16 b show the phase images and sections of spheroids grown fromcells plated into Matrigel after 7-10 days in culture. Shmac 2 and P4E6cells formed large spheroids in the absence of stroma, sectioningrevealed them to be mutilayered. Stromal co-culture reduced their sizeand led to the loss of lumen. Shmac 5 and 6 cells also formed smallerspheroids in the presence of stroma, though the differences were lessapparent. Shmac 5 cells formed acinus-like spheroids both with andwithout stromal co-culture and had predominantly single layers ofepithelia. Examination of several sections indicated the epithelia grownin the presence of stroma were predominantly columnar or cuboidal,whereas culture in the absence of stroma produced cuboidal or stratifiedcells. Examination of single epithelia within Shmac 5 spheroids grownwith stroma indicated the cells showed luminal polarization ofmicrovilli, secretory vesicles and Golgi (FIG. 17). Golgi were notablyextensive throughout the cytoplasm. Nuclei were mainly basal inposition. In addition, desmosomal-like junctions were visible betweenadjacent cells. The lumen showed very little cellular debris or necroticcells. Shmac 5 cells grown in the absence of stroma could also exhibitpolarization of intracellular organelles, though this was lessfrequently observed. Shmac 6 cells produced small spheroids which showedno evidence of lumen.

EXAMPLE 10 Phenotype of Shmac 5 Matrigel Spheroids

Shmac 5 Matrigel spheroids were further investigated byimmunocytochemical analysis, results are summarized in table 3. Inparticular, evidence of cellular polarization was examined (FIG. 18).The Shmac 5 spheroids demonstrated a phenotype very similar to primaryepithelial cell spheroids co-cultured with stroma {25970}. The outercells of the spheroid stained for basal cytokeratins, whilst the innercells stained for luminal cytokeratins or co-localised for both.Androgen receptor expression was now apparent throughout the cytoplasmof all the cells in the spheroid, and accasionally in the nucleus. Therewas little localisation of PSA expression to the lumen and little basalexpression of β1 integrins. However, CD44 did localize to the basalsurface of the spheroid. PSMA expression was cytoplasmic or membranespecific and E-cadherin was cytoplasmic or found at cell:cell membranesindicating it was functional. EXAMPLE 11

Immortalisation or Extended Life Span of E6 Transformed ProstateEpithelial Cells?

Introduction of the E6 gene into a primary prostatic cell culture servesto extend the lifespan of the cells. For prostatic epithelium, anyextension beyond passage 3-4 represents an extension of life-span. Inthe initial stages, the cells are genetically stable and resemble theoriginal culture in morphology (see FIG. 19). The cells are NOT howeverimmortal at this stage, and require to pass through a crisis for fullimmortalisation to occur. After the crisis period, the cells are stillepithelial in morphology, although certain chromosomal rearrangementswill have occurred.

The extended lifespan cells, produced after the first retroviralinfection, are genetically stable, and behave in a very similar way tothe original primary cells in most of the biological assays for up to 12population doublings. If extremely large cell numbers are not required,then the extended life span cells are preferable. To maintain thesecells, a proportion of the culture should be preserved by standardcryo-preservation at every passage, particularly while the cells areproliferating.

EXAMPLE 12 Passage Numbers

For most of the E6 extended life span cultures, at least 25 passages arepossible after introduction of the E6 gene and selection of cell clones.This is unpredictable, and may depend on the age of the patient. We haveseen no relationship to the tumorigenic phenotype however. Almostinevitably, after up to 30 passages the epithelial cells enter a crisis,and a prolonged GO phase. The essential requirement at this point is thepatience to maintain the cultures for periods up to 6 weeks, feedingessentially static and apparently dead cells. Spontaneously immortalisedcells emerge from this crisis infrequently, frequently resembling theoriginal culture, but with a less epithelial phenotype (expression ofvimentin—a stromal marker- is sometimes upregulated compared to theoriginal culture)

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1. An in vitro method for the formation of prostate-like acinicomprising: i) providing a cell culture vessel comprising: a) prostatederived epithelial cells; b) a collagen-based cell support matrix towhich the cells in (a) can attach and proliferate; c) cell culturemedium with supplemented serum, a stromal fraction and a suitable ratioof the hormones oestrogen and dihydrotestosterone, or functionalderivatives thereof; ii) providing conditions which promote the growthand differentiation of said prostate derived cells in said vessel.
 2. Amethod according to claim 1 wherein said stromal fraction is provided ina separate insert in said cell culture vessel, but in liquid contactwith the other components of the supplemented cell culture medium, whichallows said cells contained in said stromal fraction to proliferate butprevents cell contact with the prostate derived epithelial cellscontained in said vessel.
 3. A method according to claim 1, wherein saidprostate cells are human epithelial cells.
 4. A method according toclaim 3 wherein said epithelial cells are derived from prostate glandswhich have been maintained as explants for at least 7 days.
 5. A methodaccording to claim 1, wherein said prostate derived cells arenon-cancerous.
 6. A method according to claim 1, wherein said prostatederived cells are cancerous.
 7. A method according to claim 1, whereinsaid epithelial cells are primary prostate epithelial cells.
 8. A methodaccording to claim 1, wherein said prostate derived epithelial cells aregenetically engineered by recombinant techniques.
 9. A method accordingto claim 8 wherein said prostate derived cells are transformed with anoncogene.
 10. A method according to claim 9 wherein said oncogene is aviral oncogene.
 11. A method according to claim 10, wherein said viraloncogene is selected from the group consisting of: Human Papilloma Virus(HPV) E6 and E7 oncogenes, and SV40 T antigen.
 12. A method according toclaim 1, wherein serum is provided at between about 0.5%-4% (v/v).
 13. Amethod according to claim 12 wherein serum is provided at about between1%-3% (v/v).
 14. A method according to claim 13, wherein serum isprovided at about 2% (v/v).
 15. A method according to claim 1, whereinoestrogen is provided at about 10 ng/ml and dihydrotestosterone at about10⁻⁷M.
 16. A cell culture composition comprising a collagen based cellsupport; stroma, oestrogen and dihydrotestosterone.
 17. A compositionaccording to claim 16 wherein oestrogen is provided at about 10 ng/mland dihydrotestosterone at about 10⁻⁷M.
 18. A prostate like-acinusformed by the method according to claim
 1. 19. A cell derived from theprostate acinus formed by the method according to claim
 1. 20. A methodto identify agents capable of inhibiting the proliferation of cancerousprostatic cells comprising: i) providing culture conditions and at leastone cancerous acinus according to claim 18; ii) adding at least oneagent to be tested; and iii) monitoring the anti-proliferative activityof the agent with respect to the cells comprising the cancerous acinus.21. A method to identify agents capable of inhibiting the motility ofcancerous prostatic cells comprising: i) providing culture conditionsand at least one cancerous acinus according to claim 18; ii) adding atleast one agent to be tested; and iii) monitoring the motility of cellscomprising the cancerous acinus.
 22. An agent identified by the methodaccording to claim
 21. 23. A method to identify markers of prostate celldifferentiation comprising: i) providing a prostate acinus according toclaim 18; and ii) determining the presence of a RNA or protein moleculeindicative of prostate cell differentiation.
 24. A method to identifymarkers of prostate cell transformation comprising: i) providing aprostate acinus according to claim 18 and ii) determining the presenceof a RNA or protein molecule indicative of prostate cell transformation.25. An in vitro method to analyse the development of cancerous prostaticcells from normal prostatic cells comprising exposing acini according toclaim 18 to at least one agent capable of inducing prostatic celltransformation.
 26. A method according to claim 25 wherein said normalprostatic cells are transformed with an oncogene.
 27. A method accordingto claim 26 wherein said oncogene is a viral oncogene.
 28. A methodaccording to claim 27 wherein said viral oncogene is a human papillomavirus oncogene. 29-30. (Canceled)
 31. An agent identified by the methodaccording to claim 20.